An adult with cystathionine beta-synthase deficiency, camptodactyly-arthropathy-coxa vara-pericarditis syndrome, and deafness: A case report

Abstract Massive sequencing platforms allow the identification of complex clinical phenotypes involving more than one autosomal recessive disorder. In this study, we report on an adult patient, born to a related couple (third degree cousins), referred for genetic evaluation due to ectopia lentis, deafness and previous diagnosis of juvenile idiopathic arthritis. He was biochemically diagnosed as having Classic Homocystinuria (HCU); Sanger sequencing of the CBS gene showed the genotype NM_000071.2(CBS):c.[833T>C];[833T>C], compatible with the diagnosis of pyridoxine-responsive HCU. As he also had symptoms not usually associated with HCU, exome sequencing was performed. In addition to the variants found in the Sanger sequencing, the following variants were identified: NM_001256317.1(TMPRSS3):c.[413C>A];[413C>A]; and the NM_005807.6(PRG4):c.[3756dup]:[3756dup], confirming the diagnosis of autosomal recessive nonsyndromic deafness and Camptodactyly-Arthropathy-Coxa Vara-Pericarditis Syndrome (CACP), respectively. Genomic analysis allowed the refinement of the diagnosis of a complex case and improvement of the patient’s treatment.

The concept of multilocus pathogenic variations (MPV) is relatively new, corresponding to the occurrence of two or more monogenic disorders in the same individual, which leads to complex phenotypes difficult to identify only by clinical or biochemical tests (Baltaci et al., 2021;Correia-Costa et al., 2022;Herman et al., 2022).Since the advent of genome/exome sequencing, MPVs have been identified in 1.4% to 7.2% of patients with rare diseases (Yang et al., 2013(Yang et al., , 2014;;Posey et al., 2017;Correia-Costa et al., 2022).
For autosomal recessive disorders, homozygous MPVs can reach up to 29% of patients in populations that present high rates of consanguineous marriage (Mitani et al., 2021).
We report herein an unprecedented case of an adult patient with ectopia lentis, juvenile idiopathic arthritis (JIA), deafness, and psychiatric disorder who ended up being diagnosed with three monogenic diseases.This study was approved by the Local Research Ethics Committee and the patient provided written informed consent to the analyses and publication of the results.
A 33-year-old male patient was sent to genetic evaluation due to multisystemic symptoms.At physical exam, he presented weight at p90, height between p50-90, no facial dysmorphisms, contractures in hands (camptodactyly) and elbows, and genu valgum.He was the first-born of a related Donis et al. 2 couple (the parents were third cousins), with no family history of genetic diseases (Figure 1).
At the age of three months, he presented contractures in his hands, being submitted to surgical correction at one year of age.At the age of two, he presented edema in his knees and at six, he was evaluated by a rheumatologist and diagnosed with JIA.At that time, inflammatory markers were normal and treatment with methotrexate and corticosteroids was started.His psychomotor development was normal, and he developed bilateral hearing loss at age 9.He had right and left total hip arthroplasty at the age of 20 and 21, respectively, considered at that time to be secondary to the prolonged corticosteroid treatment; surgery for ectopia lentis at 23 years of age; and a single seizure at age 26.No acetabular cyst was detected (Figure 2).Echocardiogram performed at age 29 showed mild aortic insufficiency.After a few years, he started having auditory hallucinations, but no definitive psychiatric diagnosis was made.A hand X-ray was performed at age 37 and showed periarticular osteopenia, deformity with subluxation of the bilateral proximal interphalangeal joints, bone proliferations along the bilateral ulnar radio articulations, pseudocysts in the styloid processes, and tenuous images with calcic attenuation projected laterally in the region of the triangular fibrous cartilage (Figure 3).
Targeted genetic analysis (Sanger sequencing of the CBS gene) confirmed a homozygous pathogenic variant in CBS NM_000071.2:c.[833T>C]:[833T>C](p.(Ile278Thr)), which is located at exon 8, and associated with pyridoxine responsiveness (Figure 4a).However, HCU alone did not explain the JIA diagnosis; hand and elbows contractures; and hearing loss.Thus, exome sequencing was performed (exon capture with Nextera Exome Capture followed by massive parallel sequencing with Illumina HiSeq, using the GRCh37 version of the human genome as reference).In addition to the pathogenic variant in CBS, the exome sequencing revealed a homozygous pathogenic variant in TMPRSS3 NM_001256317.1:c.which is located at exon 10, related to Camptodactyly-Arthropathy-Coxa Vara-Pericarditis (CACP) Syndrome (OMIM 208250; Figure 4b-d).At the time all diagnoses were made, his medication regimen was: pyridoxine 500 mg/ day, folic acid 5 mg/day, valproic acid 750 mg 3 times/day, olanzapine 15 mg/day, phenobarbital 100 mg/day, escitalopram 10 mg/day, and methotrexate 5 mg twice a month.Once the JIA diagnosis was dismissed, methotrexate was stopped.All his siblings were evaluated biochemically and clinically, and had normal levels of tHcy and methionine, normal hearing, and normal musculoskeletal exam.A limitation of this study is that the parents and siblings of the proband were not genetically  investigated to confirm or exclude the carrier status.Genetic counseling was provided to the family.
Exome sequencing may be used if, despite consistent phenotypic features, a single genetic locus cannot explain the full phenotypic spectrum of a condition (Karaca et al. 2018).However, the large amount of data generated requires a careful analysis to detect significant variants for patient's disorders.In this sense, a detailed description of the patient's clinical manifestations is essential for the correct diagnosis.Data suggest exome sequencing results in diagnoses of 30-50% of patients tested and around 4.6-7% of patients are diagnosed with two independent monogenic conditions (Posey et al., 2017;Yang et al., 2013Yang et al., , 2014;;Ferrer et al., 2019).
After the genetic evaluation, the first clinical hypothesis and diagnosis in this patient was HCU, with a pyridoxineresponsive phenotype (Shih et al., 1995).HCU is a rare autosomal recessive inborn error of metabolism, due to impaired conversion of Hcy to cystathionine leading to hyperhomocysteinemia.There is a wide range of clinical manifestations, varying from severe childhood-onset multisystem disease to asymptomatic individuals until adulthood.Main findings are ectopia lentis, osteoporosis, 'marfanoid' habitus, learning difficulties, psychiatric disorders and predisposition to thromboembolism (Morris et al., 2017).
HCU is typically classified into three phenotypes: pyridoxine-responsive, partial pyridoxine-responsive, and pyridoxine-unresponsive.Individuals with the responsive form usually have milder phenotypes and are treated with pyridoxine (vitamin B6) and folic acid.On the other hand, unresponsive patients may require, in addition to pyridoxine and folic acid, a methionine restricted diet with methionine-free formula supplementation, and betaine.Most of the individuals with atypical or attenuated phenotypes remain undiagnosed for several years (Morris et al., 2017).The treatment prevents the occurrence of thromboembolism, which is the main cause of death of these patients.The p.(Ile278Thr) variant in the CBS gene has been described as the most widely dispersed variant, and homozygous patients present a mild phenotype, usually responsive to pyridoxine treatment (Weber Hoss et al., 2020).
In addition, the patient had some atypical findings such as hearing loss and JIA, that could not be explained by HCU.After performing exome sequencing to clarify the phenotype, he was diagnosed with two other autosomal recessive diseases: CACP syndrome and recessive nonsyndromic deafness.Furthermore, the variant in the TMPRSS3 gene is just 675 Kb from the variant present in the CBS gene, on chromosome 21, so both are likely to be segregated in conjunction.Thus, it is important to point out the occurrence of parental consanguinity as a risk factor for the autosomal recessive diseases in the offspring.
Consanguinity means that two people are descended from the same ancestor.In practice, the likelihood of sharing carrier status for a rare autosomal recessive disease is greater for related couples than for unrelated individuals.However, this increased risk does not apply to offspring if the common ancestor of the couple is more remote than second-degree cousins (Fareed and Afzal, 2017).On the other hand, it has been estimated that every individual carries more than 20 pathogenic variants associated to autosomal recessive diseases, but these estimates may vary in different populations considering aspects such as founder effect, endogamy, nonrandom mating among others.For example, in Northern Europe the estimated minimum risk of an unrelated couple having a child with an autosomal recessive disorder is 1 in 628 pregnancies (Antonarakis, 2019).In the case presented herein, the parents referred to be third cousins.Therefore, the theoretical probability of the couple having a child with at least one autosomal recessive disorder is the same as that presented by the general population.However, the patient is homozygous for all the rare variants found (autosomal recessive patients born to non-related couples are usually compound heterozygotes, unless the variant is relatively frequent), which reinforces the importance of the consanguinity in this case.Besides that, the haplotype including pathogenic variants in CBS and TMPRSS3 genes can be explained by the relationship between his parents.As the history of the family was obtained by interview, we cannot exclude a mistake on the reported consanguinity, although the occurrence of three monogenic diseases in the same individual can also be random or explained by other factors.For example, since the variants found have not been searched in both parents, we cannot exclude non-maternity and/or non-paternity.
Pathogenic variants in TMPRSS3 have been implicated in prelingual and postlingual hearing impairment.There is a genotype-phenotype correlation where loss-of-function variants cause the most severe phenotype, while missense variants are associated with a milder form of hearing impairment (Gao et al., 2017).The p.(Ala138Glu) variant has been associated with severe-to-profound hereditary highfrequency sensorineural hearing loss before 15 years of age.Furthermore, patients carrying this variant have poor cochlear implant performance, which is the gold-standard treatment for this disorder (Chen et al., 2022).Our patient had postlingual hearing impairment at nine years such as described in patients from other studies (Weegerink et al., 2011).
Proteoglycan 4 (PRG4) is expressed in chondrocytes of the superficial zone and is involved in lubricating and protecting cells in the surfaces of joints and tendons as well as in non-skeletal tissue including liver and pericardium (Albuhairan and Al-Mayouf, 2013).Pathogenic variants in PRG4 identified on chromosome 1q25-q31 cause the CACP syndrome, characterized by congenital or early-onset camptodactyly, childhood-onset non-inflammatory arthropathy of large joints -such as elbows, hips, knees, and anklessynovial hyperplasia, progressive coxa vara deformity and non-inflammatory pericardial or pleural effusion (Albuhairan and Al-Mayouf, 2013;Vutukuru and Reddy, 2016).Due to overlapping symptoms, CACP may be misdiagnosed as JIA, which is treated with immunomodulators (Kisla Ekinci et al., 2021).Importantly, the specific musculoskeletal features of CACP syndrome do not respond to this type of medication, therefore affected individuals should be treated with pain medications and physical therapy (Albuhairan and Al-Mayouf, 2013).Some clinical features that may help to differentiate CACP from JIA are the presence of coxa vara, positive family history, normal inflammatory markers, absence of inflammation in synovial aspirates or synovial biopsies, and typical imaging findings including the presence of intraosseous cysts (Marcelino et al., 1999;Madhusudan et al., 2016).
Providing patient with the correct diagnosis allowed us to withdraw methotrexate which is known to cause serious and long-term severe adverse effects such as predisposition to infections, lung and skin problems, dizziness, and gastrointestinal issues as well as tailor the physical therapy and other medications to treat his diseases.Besides that, methotrexate can increase the tHcy levels.
Here, we report the success of exome sequencing in diagnosing an individual with three autosomal recessive disorders.This case highlights the importance of further investigations when patient's symptoms are not explained by a single disease.An accurate diagnosis of the coexistence of multiple conditions can allow for the cessation of ineffective and potentially harmful treatments.

Figure 1 -
Figure 1 -Family History.The family pedigree representing six generations.Subject IV-4 died of acute myeloid leukemia.Subject V-2 died at 5 months (skin lesions/meningitis/hospitalized for 3 months).No other family member presents clinical manifestations of Classic Homocystinuria or Camptodactyly-Arthropathy-Coxa Vara-Pericarditis Syndrome or autosomal recessive nonsyndromic deafness.

Figure 2 -
Figure 2 -Bilateral hip and knee X-ray showing osteonecrosis of the left femur head and right prosthesis (A) and osteopenia (B).

Figure 3 -
Figure 3 -Bilateral hand X-ray.Pseudocysts are seen in the styloid processes (arrows) and bilateral deformity with subluxation of proximal interphalangeal joints (arrowheads).